TITLE: Probing the mechanistic basis for T cell fate decisions. CD4+ T cells use highly sensitive and specific modular biosensors to survey the body for microbial pathogens or tumors. The TCR is the chief mediator of this behavior. It surveys the contents of MHC on antigen presenting cells for peptides (pMHC) derived from microbial pathogens or tumors, and relays pMHC-specific information across the cell membrane to the ten immunoreceptor tyrosine-based activation motifs (ITAMs) of the associated CD3?? , CD3??, and CD3?? signaling modules. CD4 recruits Lck to the TCR-CD3 complex upon concurrent binding of MHC. The quantity and quality of ITAM phosphorylation by Lck provides the base set of instructions that inform CD4+ T cell fate decision. Yet, how the individual subunits of the TCR-CD3-pMHC-CD4 macro-complex fit and work together to drive CD4+ T cell fate decisions remains to be fully defined. Our working hypothesis is that these molecules operate on a similar mechanistic principle to less complex receptor systems, such as cytokine receptors, whereby receptor-associated intracellular signaling enzymes and their substrates are held in a spatial relationship that represents ?off?; pMHC-engagement and reciprocal extracellular interactions between the TCR-CD3 complex and CD4 then positions the intracellular signaling domains in the appropriate spatial relationship for a sufficient duration to initiate and potentiate signaling. Our overarching goal is understand the inner workings of this complex molecular machinery so that we can modify or imitate its form and function to design novel modular biosensors with unique therapeutic functions. During the previous funding period we built multiple experimental platforms to study the spatial relationship between the juxtamembrane (JM) regions of the TCR-CD3 subunits. These allowed us to report the identification of a mechanical switch that relays pMHC-specific information from the TCR-pMHC interface across the T cell membrane to the cytosolic juxtamembrane regions of the CD3?? signaling module. In addition, we performed the first experimental analysis of the architecture of the TCR-CD3-pMHC-CD4 macrocomplex and found that the CD4 JM region is proximal to the CD3 heterodimers, while CD3?? resides on the opposite side of the TCR. We also identified highly conserved residues in the TMD and extracellular domains of CD4 that are important for CD4's Lck-independent and Lck-dependent functions. Finally, we obtained functional evidence for TCR- intrinsic specificity for MHC that we interpret as evidence for MHC scanning. The goals for this renewal application are to deconstruct the molecular mechanisms by which the TCR-CD3-pMHC-CD4 macrocomplex operates and characterize the consequences of these mechanisms in vivo. Our work will yield fundamental insights into the key determinants of CD4+ T cell fate decisions and provide a blueprint for the development of novel modular biosensors with translational potential.